Attention is being given to the importance of analyses and measurements carried out at or near the site where the analyses and measurements are required (hereinafter referred to as “POC (point of care) analyses and the like” collectively), such as analyses for bedside diagnosis (POC analyses) in which a measurement required for medical diagnosis is carried out near a patient, analyses of hazardous substances in rivers and wastes carried out at sites such as rivers and wastes treatment plants, and contamination tests at individual sites for cooking, harvest and importation of foods. And, in recent years, development of measurement methods and apparatuses suitable for these POC analyses and the like has been considered as an important matter.
In these POC analyses and the like, it is required that analysis be able to be carried out conveniently, for a short time and inexpensively, and particularly in analyses for medical diagnosis, reduction in analysis time and reduction in the amount of sample required for analysis to a very low level are important challenges to be addressed.
In the blood test, for example, if the amount of sample required for analysis is very small, the amount of blood to be collected can be reduced, thus alleviating burdens on patients, and increasing the possibility of application to at-home medical care such as self analysis by self blood-collection. Also, if the amount of sample required for analysis is very small, then the amount of reagent for use in analysis can also be reduced to a very low level, thus making it possible to reduce costs involved in the analysis. In addition, studies are continued for reduction in the amount of sample to a very low level and reduction in analysis time, because the goal of reducing the amount of wastes can be thereby achieved at the same time.
In the case of qualitative analysis in which a determination is made by visual observation or the like, methods in which a sample such as blood, urine and polluted water is directly contacted with a test paper impregnated with an analyzing reagent are widely used. However, in the case of quantitative analysis of blood biochemistry in which the enzyme activity in the blood and the amount of substance to be measured are analyzed, quantifiability is required, and therefore most of the methods currently used in medical practice are such that an reagent solution, buffer and the like set in an analyzer are mixed and quantitatively reacted with a sample weighed and taken in a cuvette or test tube in the analyzer using an automatic pipetter or the like to make a detection with a detection device.
However, in the case of these methods, the reagent solution, buffer and the like set in the analyzer should be replenished as appropriate. Also, there are possibilities that troubles occur such as spilling of liquid, clogging of the nozzle of the automatic pipetter and contamination due to insufficient cleaning, and analysis operators, particularly doctors and nurses in the case of POC analyses in the medical field, may significantly be burdened.
For solving the above problems, a measuring system of all-in-one-type is desired in which a required reagent solution and buffer and the like are provided in the analyzing cartridge. As one example, a method in which an internal bag containing therein the reagent and the like is encapsulated in a bag containing therein the sample, and the above described internal bag is pressed by hand to break the same, thereby mixing and reacting together the sample and the reagent and the like, and then a qualitative determination is made by visual observation is disclosed in Japanese Patent Laid-Open No. 8-160031. However, such a method is not suitable for quantitative analysis in a field of blood biochemistry and the like requiring a high level of accuracy.
Ideas of containing required reagents in the cartridge in advance are also disclosed in the apparatus structures in Japanese Patent Laid-Open No. 2-87067 and Japanese Patent Laid-Open No. 2-88970. In this case, the reagent solution and the like are contained in a small bag, and the bag is broken, whereby the reagent solution and the like are leaked into the cartridge. Also, for the method for feeding a liquid, a method using centrifugal force is adopted.
In the case where the amount of sample is large, and the amount of the reagent solution and the like for analysis is also large ranging from a few hundred μl to a few ml, the solution and the like can be encapsulated in the above described bag easily and inexpensively, and the above described method can be used without any problems. However, when the amount of sample is as small as a few μl or so, a small amount of reagent solution and the like consistent with the small amount of sample should be encapsulated in a small bag, and the bag should be broken to leak the contents, and such operations are extremely difficult with the above described method.
In addition, a technique in which a liquid reagent and the like are encapsulated in a small container provided in the cartridge, and the breakable portion (breakable seal) of the small container is broken with a pointed object or the like to let the liquid reagent leak through, and mix and quantitatively react with the blood sample is also disclosed. For example, in the cartridge for measurement of hemoglobin Alc (HbAlc) of Japanese Patent No. 2790359 (Boehringer Mannheim K.K.), gravity and capillarity force are used for feeding of liquids, the breakable seal is broken to let the liquid reagent encapsulated in the cartridge leak through into a dilution vessel having a predetermined volume, where the liquid reagent is mixed quantitatively with a sample in the capillary having a predetermined volume to carry out a measurement of hemoglobin (Hb) after hemolysis and a subsequent measurement of HbAlc with latex beads.
Also, in the cartridge for an automatic analyzer disclosed in International Patent Publication WO 93/25889, a solid reagent is encapsulated in the cartridge, and a diluent and a liquid reagent are supplied from the outside of the cartridge. And, the breakable seal is broken by a pointed object such as a nail, whereby the solid reagent is mixed with the diluent and dissolved therein, and is mixed with the blood sample together with the liquid reagent.
In addition, a cartridge having a structure in which a liquid reagent is pushed out by air through a “bursting channel” at the exit of the chamber with the liquid reagent encapsulated therein, and an air bubble generated at this time is trapped in a chamber (the bubble is not pushed out from the chip) is disclosed in International Patent Publication WO 99/52633 (Lumenal Technology Co., Ltd.).
For a method using a breakable seal like this, as in the case of the aforesaid method using a bag, if the amount of the encapsulated liquid sample is large ranging from a few hundred μl to a few ml, the liquid sample can be encapsulated easily and less expensively, but if the amount of the sample is as small as a few μl or so, the liquid sample becomes very small accordingly so that it will be extremely difficult to incorporate the breakable seal in the aforesaid small container, to inject and encapsulate a very small amount of liquid sample, to break the breakable seal, to take out the liquid sample and so on.
Also, techniques in which the liquid reagent (reagent solution) and the buffer and the like are encapsulated in the cartridge, and the liquid is fed to mix and react the same together using a centrifugal force also include techniques in Japanese Patent Publication No. 4-53384B and Japanese Patent Laid-Open No. 8-62225A.
In the methods as described above, because the reagent and the buffer are encapsulated in the cartridge in liquid states, and a mechanism to take out the liquid such as a mechanism to break the seal is required, the cartridge needs to have a very complicated structure. Also, for encapsulating the reagent in a liquid state and taking out the reagent into the reaction channel, a reagent of a few ten to a few hundred μl or more is required, and thus the cost for the reagent is increased.
In addition, in most of the above described methods, centrifugal force is used for feeding the liquid. In this method, the direction in which the liquid is fed is limited to one direction extending outwardly from the center of rotation, and rotation should be started/stopped accurately in the on/off of the feeding of liquids and so on, resulting in complicated designs for the feeding of liquids and channels. In addition, there is also a disadvantage that the analyzer with which the cartridge is equipped to carry out measurements is complicated in structure and expensive. Also, in any case, since the thickness of the capillary is in the order of millimeters, and thus a lager amount of sample is required, and the amount of reagent is increased in association therewith, the cost involved in analysis is no longer reduced, along with the aforesaid increase in the amount of liquid reagent encapsulated.
On the other hand, a method is disclosed in which a freeze-dried solid reagent is placed in the cartridge, the blood sample is diluted with a dissolving diluent encapsulated in the cartridge, and the above described solid reagent is dissolved in the diluted sample solution, and is reacted therewith to carry out analysis (Published Japanese translation of PCT international publication for patent application No. 10-501340, Published Japanese translation of PCT international publication for patent application No. 9-504732 and the like).
In this method, the solid reagent is placed in chambers on the circumference located at the end of the channel in the cartridge, and the diluted sample is flown into each chamber to dissolve and react with the solid reagent, thus bringing about a change in absorbance. Also, since the liquid is fed by means of centrifugal force, the direction in which the liquid is fed is limited to the direction of the centrifugal force, namely a direction extending outwardly from the center of the circle of the circular cartridge.
As described above, for the detection reaction, only one reaction of one reagent composition can be carried out because the solid reagent is located at the end of the channel. Therefore, for some detection items, a reaction and reagent composition different from those of detection reactions specified in recommendations defined by academic societies and governmental agencies, which are conducted in medical laboratories and clinical laboratories of hospitals, must be employed. That is, in those recommendations, two or three types of reagent solutions are often used when one substance is detected (for example, described in Summary of Clinical Examination Process (written by Izumi Kanai, Kanahara press. 1998.), and if one type of reagent is used, correlation with previous examination data may be poor, and quantitation itself may be difficult to carry out.
Also, air in the above described chamber is pushed out by the inflow of a liquid, but since the chamber is located at the end of the channel, the air cannot be discharged from the cartridge, and should be moved to other location in the cartridge. Therefore, a design is made so that the above described air is let out through the upper space of the channel into which a liquid is flown. In other words, there is a disadvantage in terms of channel that the liquid and gas are flown in opposite directions in a very narrow channel.
A technique in which droplets of reagent solution are dropped in liquid nitrogen to be frozen in spherical forms, and are dried directly under a reduced pressure for dissolving in a short time the reagent placed in the chambers on the circumference is disclosed in International Patent Publication WO 93/04195, Published Japanese translation of PCT international publication for patent application No. 10-501340A and Published Japanese translation of PCT international publication for patent application No. 9-504732A.
In these documents, it is also disclosed that polyethylene glycol, myoinositol, polyvinyl pyrolidone, dextran, sodium cholate, mannitol, albumin or a mixture thereof is used as an adjuvant for forming droplets in good quantitation and enhancing solubility. Furthermore, it is described that glycerol is added in Example 3 of International Patent Publication WO 93/04195 (ALP Measurement Reagent), but its object is not been clarified.
A method is also disclosed in which the solid reagent deposited in the cartridge is dissolved in plasma being just a sample without using a sample dissolving liquid to carry out analysis. It is disclosed in, for example, Trade Name: Twinkle® of Nippon Medi-physics Co., Ltd., Japanese Patent Laid-Open No. 9-196920 (Immune Item), Japanese Patent Laid-Open No. 8-114539 (Biochemical Item), Japanese Patent Laid-Open No. 9-196739 (Dissolving Liquid Tip detection) and Japanese Patent Laid-Open No. 9-138195 (Analysis by Transmittance Optical measurement of Porous Material).
In this method, the solid sample is dissolved in plasma itself while a reaction is carried out at the same time, but it is not easy to dissolve uniformly a solid reagent using 100% plasma in a very short time. Also, there is the problem of vulnerability to contaminants (inhibiting substance) inhibiting analysis because the plasma is not diluted.
In these disclosed techniques, a method in which a liquid is suctioned under a reduced pressure from the end of the channel is employed as a principal method of feeding the liquid. In other words, a plurality of openings leading to the outside of the cartridge are provided in the direction of flow at a predetermined interval in one channel, and the feeding of the liquid is controlled by opening/closing the openings outside the cartridge. However, in this method of feeding the liquid, there is a disadvantage that the flow of the liquid can be controlled only linearly. These openings do not have the function of hydrophobic vents, but play a role as air release valves capable of reducing a pressure in the areas upstream of the openings by closing the openings.
In addition thereto, methods in which the reagent is deposited through the channel include those disclosed in U.S. Pat. No. 5,478,751 Specification (Abbott Co., Ltd.), Japanese Patent Laid-Open No. 3-223674 (Mochida Pharmaceutical Co., Ltd.) and U.S. Pat. No. 5,147,607 Specification (Mochida Pharmaceutical Co., Ltd.), and these method are not different from the above described methods in a sense that the sample is contacted with the reagent of high concentration and diluted in succession although substances to be flown through the channel are not necessarily 100% plasma. Those methods are used in the case where a solid phase antibody (antigen) is contacted with the sample as in immune-detecting reaction, but is not suitable for blood biochemical examination based on the reaction in a homogeneous system and examination of hazardous substances in the environment.
In addition, methods in which a filter paper or the like is impregnated with the analyzing reagent, the reagent is contacted with the blood sample, and the plasma is moved on the filter paper using the capillary force and gravity to carry out analysis have gone into actual use. These methods include, for example, those described in Spotchem® (trade name) manufactured by Kyoto Daiichi Kagaku Co., Ltd., Drychem® (trade name) manufactured by Fuji Photo Film Co., Ltd., Reflotron® (tradename) manufactured by Boehringer Mannheim K.K. or U.S. Pat. No. 5,212,065 (International Diagnostic System).
The so called analyzing filter paper based cartridges based on dry chemistry are convenient because they enable quantitative reaction while the reagent solution and buffer do not need to be added from the outside. However, the amount of required blood sample is large, namely about 10 μl for each item of analysis, which means that one hundred and a few ten μl of blood is required in the case of measurements for ten items or more that are usually conducted in the blood biochemical examination. Also, in association therewith, the amount of reagent used to impregnate the filter paper or the like is increased. Also, because the sample is contacted and reacted in succession with the reagent with which the filter paper or the like is impregnated, the type of analysis reactions is limited, and some of them are different from the aforesaid recommended methods defined by academic societies.
In addition, since the sample is not diluted, possibilities of being adversely influenced by contaminants in the sample are increased. Also, for the most part, one cartridge is required for each item of analysis, and only the method of Kyoto Daiichi Kagaku enables a plurality of analyses to be carried out at the same time, but in this method, essentially a plurality of cartridges each being used for one item of analysis are placed on the same strip, allowing only six items to be analyzed at the maximum.
On the other hand, in contrast to the above described methods using dry chemistry, the technique of μTAS (micro total analysis system) to carry out micro analysis has been developed. In the μTAS, for reducing the amount of any sample as well as blood to a very small level, a chip of a few to ten centimeter square with grooves provided on the surface of glass or silicone is used, and the reagent solution and sample are flowed in the groove to carry out separation and reaction to analyze a very small amount of sample (Japanese Patent Laid-Open No. 2-245655A, Japanese Patent Laid-Open No. 3-226666A, Japanese Patent Laid-Open No. 8-233778A, Analytical Chem. 69, 2626-2630 (1997) Aclara Biosciences, etc.). For this technique, there is an advantage that the amount of sample, the amount of reagent required for detection, the amount of wastes from consumable products used in detection and the amount of waste liquids are all reduced, and time required for detection can be shortened.
The inventors have also submitted applications related to the μTAS such as Japanese Patent Application No. 10-181586 specification (“Mixing analysis apparatus and mixing analysis method”), Japanese Patent Laid-Open No. 2000-2675A (Japanese Patent Application No. 10-181587 Specification “Capillary Photothermal Converting analysis apparatus”), Japanese Patent Laid-Open No. 2000-2677A (Japanese Patent Application No. 10-167603 Specification “Analysis apparatus”), and International Patent Publication WO 99/64846 (International Patent Application PCT/JP99/03158 Specification).
If the technique described in these specifications is used, the amount of reagent solution required for analyzing one item of blood biochemistry can be reduced to as small as about 10 nl, and the amount of sample required can be reduced to about 1 to 0.1 nl (1000 to 100 μl) with the detection time being about 10 seconds (About 1 μl of buffer for feeding the liquid entirely is required in addition thereto. Also, about 10 nl of sample may be required for 10 minutes if the sample is continuously fed).
However, in μTAS techniques that are now widely known, separation, mixture, reaction and detection are carried out within the chip (cartridge), but the reagent solution required for reaction is supplied from the outside of the chip (cartridge). Those techniques include, for example, those of prior arts related to the aforesaid μTAS, Proceedings of the μTAS '98 Workshop, held in Banff, Canada, 13-16 Oct. 1998. Editors: D. Jed Harrison and Albert van den Berg, Kluwer Academic Publishers and the like, and techniques of DNA analysis in a resin chip (R M Mccormick et al./Anal. Chem. Vol. 69, No. 14 (1997) 2626-2630, etc.).
These techniques are not suitable for the POC analyses and the like where simplicity is required, because a container for the liquid reagent needs to be provided outside the chip, maintenance operations for replenishing the liquid reagent, removing clogs or performing cleaning in the connection between the chip and the container are required.
On the other hand, for allowing the sample and the reagent solution to move in the chip (cartridge), air in the capillary (groove) leading to the destination to which they move needs to be discharged to the outside of the channel. At this time, it is desirable that a gas-permeable/non-liquid-permeable mechanism is provided at the end of the channel for surely keeping liquid in the chip. Otherwise, the liquid may be spilled out of the chip.
For achieving the purpose of providing this gas-permeable/non-liquid permeable mechanism, techniques in which small hydrophobic openings and hydrophobic membranes are used as vents through which not liquid but only air can be passed have been considered for rather a long time as techniques coping with a much larger amount of liquid compared to the case of the capillary.
For example, the technique of air vent from the blood in a blood processing apparatus such as an artificial dialyzer is described in Japanese Patent Laid-Open No. 57-17659A and Published Japanese translation of PCT international publication for patent application No. 9-500809A. Also, an example of providing vents in an apparatus much larger than the chip as automatic air vent filters for venting air in liquid chemicals and water that are used in factories in general is described in Japanese Patent Laid-Open No. 2-2812A. In any case, they cope with a much larger amount of liquid compared to an amount of liquid below the level of micro liters.
For those that are used as these air vents in the chip for coping with an amount of liquid below the level of micro liters, very small hydrophobic openings of about 3 μm square (HMCV (Hydrophobic Micro Capillary Vent)) are known (proceedings of the μTAS '98 Workshop, held in Banff, Canada, 13-16 Oct. 1998. Editors: D. Jed Harrison and Albert van den Berg, Kluwer Academic Publishers, p 307-310 Hydrophobic microcapillary vent for pneumatic manipulation of liquid in μTAS, Kazuo Hosokawa, Teruo Fujii, and Isao Endo, Document of Electricity Academy Workshop: Society for Study of Chemical Sensor System CS-99-1 to 12, p 19-22, Mar. 16, 1999, Teruo Fujii, Kazuo Hosokawa, Hong Jong Wook, Minoru Seki, Isao Endo et al.).
Also, a technique in which a hydrophobic membrane is provided at the end of the channel is disclosed (Affy Metrix Co., Ltd., Anderson et al., Proceeding of Transducers '97. 1997 International conference on Solid state sensors and Actuators 2C1. 02, International Patent Publication WO No. 9702357, U.S. Pat. No. 5,856,174 Specification). In this technique, the reagent solution and sample outside the chip are connected to the chip through a tube, a capillary through which the above described reagent solution and the above described sample are flown is selected with a diaphragm valve (valve opened/closed by force from the outside of the chip) provided in the chip. Then, the air in the capillary is pushed to the outside of the channel through the vents composed of a hydrophobic membrane at the end of the channel to feed the liquid. The above described vents always are opened outwardly, the feeding of the liquid is controlled by using pressure losses of the channel connected to the vents and providing pressure-relief openings with breakable seals, thus rising the problem of the structure being extremely complicated.
An object of the present invention is to solve the problems of conventional techniques as described above, and provide an analyzing cartridge as described below and a method for producing the same:
1) It is possible to prepare a reagent solution of a few μl or less in the cartridge at the time of measurement to provide the same for the reaction, which is difficult for the system in which the liquid is encapsulated in bags or chambers in the cartridge (liquid bag system), and is discharged into a reaction vessel at the time of measurement.
2) Measurements and reactions can be carried out using a very small amount of sample in the order of nanoliters to picoliters.
3) A reagent, buffer and the like required for analysis are encapsulated in the cartridge or attached thereto, and thus time and effort for management and maintenance of the reagents by an analysis operator can be alleviated or totally saved.
4) Analysis can be carried out conveniently, in a short time and inexpensively.
5) Limitations on the detection reaction are reduced, and analysis for multiple items can be carried out at the same time. Therefore, detection reactions the same as or similar to those of the recommended methods defined by academic societies, government agencies and the like can be carried out.
6) The analyzing cartridge has a simple internal structure and can thus be produced inexpensively.
7) The feeding of the liquid can be carried out accurately.
Another object of the present invention is to provide an analyzing method using the above described analyzing cartridge.
Still another object of the present invention is to provide a liquid feed control device that can be attached to the analyzing cartridge as described above, is capable of controlling the feeding of the liquid in the analyzing cartridge accurately and easily, and is inexpensive.